****< 0

****< 0.0001 compared to the control at pH 6.0; ###< 0.001 compared to the control at pH 7.4. reversed after 3 and 12 h, respectively, indicating that these inhibitors are slowly reversible. The uptake of AR-C155858 was concentration-dependent in 4T1 cells, whereas the uptake of AZD3965 exhibited no concentration dependence over the range of concentrations examined. The Ferrostatin-1 (Fer-1) uptake kinetics of AR-C155858 was best fitted to a Michaelis-Menten equation with a diffusional clearance component, (= 0.399 0.067 M, = 0.330 0.088 L/mg/min). AR-C155858 uptake, but not AZD3965 uptake, was significantly inhibited by alpha-cyano-4-hydroxycinnamic acid, a known nonspecific inhibitor of MCTs 1, 2, and 4. AR-C155858 exhibited a pattern toward higher uptake at lower pH, a characteristic of proton-dependent MCT1. These findings provide evidence that AR-C155858 and AZD3965 exert slowly reversible inhibition of MCT1-mediated L-lactate uptake in 4T1 cells, with AR-C155858 representing a potential substrate of MCT1. Ferrostatin-1 (Fer-1) > 10 nM), in studies of MCT2-transfected oocytes (30,31). AZD3965 (Fig. 1b), an analogue of AR-C155858, is an orally bioavailable inhibitor of MCT1 that is currently being investigated in a phase I clinical trial in the UK for advanced solid tumors and lymphomas (“type”:”clinical-trial”,”attrs”:”text”:”NCT01791595″,”term_id”:”NCT01791595″NCT01791595). AZD3965 shows comparable potency and specificity as AR-C155858, with 6-fold higher potency for MCT1 compared with MCT2, and no inhibition of MCT3/MCT4 at 10 M (23). AZD3965 has been demonstrated to significantly CSF3R decrease cell and tumor growth in human small-cell lung cancer and various lymphoma xenograft models that overexpress MCT1 (21C23,32,33). Open in a separate windows Ferrostatin-1 (Fer-1) Fig. 1. Chemical structure of AR-C155858 (a) and AZD3965 (b) Previous work from our laboratory has shown that inhibition by AR-C155858 was time-dependent and not rapidly reversible (34). The mode of action by AZD3965 is largely unknown and has not been studied. The objective of our current study was to further characterize the properties of AR-C155858 and AZD3965 in the murine 4T1 breast tumor cells, a relevant cell system to study MCT1 that is highly conserved across species (35), as it expresses MCT1, but not MCT2 or MCT4 (36). We evaluated the time-dependent inhibition of L-lactate uptake by AR-C155858 and AZD3965 as well as the reversibility of the inhibition. Our current study also sought to characterize the cellular uptake of AR-C155858 and AZD3965 and determine if these inhibitors are also MCT1 substrates. MATERIALS AND METHODS Chemicals and Reagents L-lactate (as calcium salt) and alpha-cyano-4-hydroxycinnamic acid (CHC) were purchased from Sigma-Aldrich (St. Louis, MO). AZD3965 and AR-C155858 were obtained from AstraZeneca, MedKoo Biosciences (Chapel Hill, NC), and Chemscene (Monmouth Junction, NJ), respectively. L-[3H] lactate was purchased from American Radiolabeled Chemicals (St. Louis, MO). Cell Culture Mouse mammary tumor, 4T1 cells were kindly provided by Dr. Elizabeth A. Repasky (Roswell Park Malignancy Institute, Buffalo, NY). Cells were maintained at 37 C in a humidified atmosphere with 5% CO2/95% air. The 4T1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 models penicillin, and 100 g/mL of streptomycin. Culture medium was changed every 2C3 days, and cells were passaged with 0.25% trypsin/EDTA. Cellular L-Lactate Uptake Studies The 4T1 cells were plated in 35-mm (diameter) culture dishes at a cell density of 2.0 105 cells/mL 2 days before the uptake study. On the day of the experiment, the culture medium was removed and cells were washed three times followed by equilibration for 20 min at 37 C with the uptake buffer made up of 137 mM N-methyl-D-glucamine, 5.4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES at pH 6.0 and 7.4. pH 6.0 was used to drive the transport by MCT1, as transport is dependent around the proton gradient. pH 7.4 represents the physiologically relevant blood pH and was used for comparison. Cells were pre-incubated with the inhibitors at 37 C and subsequently cooled to room heat (RT) for 5 min. Cells were then incubated in 1 mL of uptake buffer made up of [3H]-L-lactate and 0.5 mM of cold L-lactate for 1 min at RT. A reaction time of 1 1 min was used because it was decided that 1 min is within the time range of linear uptake of L-lactate in 4T1 cells. The uptake was terminated by aspirating the uptake buffer, followed by washing the cells rapidly three times with Ferrostatin-1 (Fer-1) ice-cold buffer. Cells were lysed in 0.5 mL of 1 1.0 N NaOH for 1 h at RT and the cell lysates were neutralized with 0.5 mL of 1 1.0 N HCl. Scintillation fluid (3 mL) was added to 400 L of cell lysate, and the radioactivity was measured by liquid scintillation counting (1900 CA, Tri-carb liquid scintillation analyzer, Packard Instrument Co., Doners Grove, IL). Protein concentrations were decided using BCA protein assay kit (BCA, Pierce.